Biological activity and interaction mechanism of the diketopiperazine derivatives as tubulin polymerization inhibitors

Abstract

Microtubules are a favorable target for development of anticancer agents. In this study, the anti-proliferative activities of plinabulin and six diketopiperazine derivatives were evaluated against human lung cancer cell line NCI-H460 and human pancreatic cancer cell line BxPC-3. The inhibition activities on these microtubules were assessed by tubulin polymerization and immunofluorescence assays. To gain insight into the interaction mechanism of the derivatives and tubulin, a molecular dynamics simulation was performed. We discovered that the diketopiperazine derivatives could prevent tubulin assembly through conformational changes. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) calculations showed that the trend of the binding free energies of these inhibitors was in agreement with the trend of their biological activities. Introducing hydrophobic groups into the A-ring was favorable for binding. Energy decomposition indicated that van der Waals interaction played an essential role in the binding affinity of tubulin polymerization inhibitors. In addition, the key residues responsible for inhibitor binding were identified. In summary, this study provided valuable information for development of novel tubulin polymerization inhibitors as anticancer agents.

Fig. 1. (a) Four microtubule binding sites: colchicine (orange), laulimalide (red), taxol (green) and vinblastine (yellow). α and β tubulins are shown in pink and blue, respectively. (b) Structures of the representative compounds.

Fig. 2. Structures of the synthesized diketopiperazine derivatives.

Fig. 3. Effects of plinabulin and compounds a–f at 5 μM on the tubulin polymerization. (a) Tubulin polymerization inhibition activities measured with time at 37 °C. (b) Comparison of IC₅₀ values (BxPC-3: green; NCI-H460: red) and tubulin inhibition rate (black) of DKP derivatives.

Fig. 4. Immunofluorescence assay of plinabulin and compounds a–f inhibition of tubulin polymerization in vitro. (a) Confocal images of DKP derivatives (5 nM) disrupting the mitotic spindles in BxPC-3 cell. (i) Nuclear (blue); (ii) tubulin (red); (iii) (i) and (ii) were overlapped. Scale bar is 50 μm. (b) Semi-quantitative analysis of the inhibition of tubulin polymerization.

Fig. 5. The reasonable binding poses of the inhibitors in tubulin. (a) Plinabulin and compound a in the pocket 5C8Y. (b) Compounds b–f in the pocket 5YL4.

Fig. 6. Structural stability of the tubulin-apo and tubulin-inhibitors complexes. (a) Backbone RMSD of dipolymer (black). (b) RMSF of side chain in α-tubulin (red) and β-tubulin (black).

Fig. 7. Structural analysis of MD simulation. Overview of the structures of (a) tubulin-apo at 0 ns (pink) and 26 ns (light green); (b) tubulin–plinabulin at 0 ns (pink) and 26 ns (light green); (c) radius of gyration of tubulin–plinabulin (red) and tubulin-apo (black) with time; (d) the pocket comparison of tubulin-apo of 0 ns (pink) and 26 ns (cyan); (e) the pocket comparison of the crystal structures of tubulin bound with plinabulin (pink) (PDB ID: 5C8Y) and with colchicine (green) (PDB ID: 1SA0); (f) the pocket comparison of 5C8Y (pink) and tubulin-apo (grey) (PDB ID: 3HKB).

Fig. 8. (a) Ligand-position RMSDs of plinabulin, compound a, compound e, and compound f; (b) structure analysis of MD simulation: tubulin–plinabulin (pink) vs. tubulin–a (green) (c) overview of the structures of tubulin–e (pink) and tubulin–f (green) after MD simulation vs. tubulin–f (grey) before MD simulation; (d) structure analysis of MD simulation: tubulin–e (pink) vs. tubulin–f (green); (e) number of hydrogen bonds: compound evs. compound f.

Fig. 9. Contributions of the key residues of plinabulin and compounds a–f. (a) α-tubulin. (b) β-tubulin.